Osteoclasts are multinucleated giant cells specialized for the removal of both the inorganic and organic phases of bone (Blair H. C., et al., J. Cell. Biol., 102:1164-1172 (1986)). The pathway(s) for degradation of the organic matrix, primarily type 1 collagen, are not well understood, although mounting evidence has implicated cysteine proteinases (cathepsins) as key enzymes in this process.
Dissolution of the hydroxyapatite mineral phase is dependent upon acidification of the subosteoclastic resorption lacuna, via the action of carbonic anhydrase II and a proton pump (Vaes, J. Cell Biol., 39:676-697 (1968); Baron et al., J. Cell Biol., 101:2210-2222 (1985); and Blair and Schlesinger, in Biology and Physiology of the Osteoclast, Rifkin and Gay, eds. (CRC Press, Boca Raton), pp. 259-287 (1992)). V-type proton pumps are multi-subunit complexes with two distinct functional domains: a peripherally-associated cytoplasmic catalytic sector that contains 70- (subunit A), 58- (subunit B), 40- and 33-kDa (subunit E) subunits (Xie and Stone, J. Biol. Chem., 263:9859-9866 (1988)), and a proton channel, which is likely composed of 116-, 39-, and 17-kDa components (Crider et al., J. Biol. Chem., 269:17379-17381 (1994)). Considerable speculation has focused on the possibility that osteoclast-specific proton pump subunits exist.
Excessive bone resorption by osteoclasts contributes to the pathology of many human diseases including arthritis, osteoporosis, periodontitis, and hypercalcemia of malignancy. During resorption, osteoclasts remove both the mineral and organic components of bone (Blair, H. C., et al., J. Cell Biol; 102:1164 (1986)).
The regulation of osteoclastic activity is only partly understood. The lack of information concerning osteoclast function is due in part to the fact that these cells are extremely difficult to isolate as pure populations in large numbers. Furthermore, there are no osteoclastic cell lines available. An approach to studying osteoclast function that permits the identification of heretofore unknown osteoclast-specific or -related DNA sequences, genes and gene products would allow identification of genes and gene products that are involved in the resorption of bone and in the regulation of osteoclastic activity. Therefore, identification of osteoclast-specific or -related DNA sequences, genes or gene products would prove useful in developing therapeutic strategies for the treatment of disorders involving aberrant bone resorption.
The present invention relates to isolated osteoclast-specific or -related DNA sequences. These sequences can be all or a portion of an osteoclast-specific or -related gene. The sequences of the present invention encode all or a portion of an osteoclast-specific or -related gene product (i.e., peptide or protein) or encode all or a portion of the untranslated portion of the genomic DNA sequence. The present invention further relates to DNA constructs capable of replicating osteoclast-specific or -related DNA. In another embodiment, the invention relates to a DNA construct capable of directing expression of osteoclast-specific or -related DNA sequences, producing osteoclast-specific or -related peptides or gene products, in a host cell.
Also encompassed by the present invention are prokaryotic or K]d cells transformed or transfected with a DNA construct comprising an osteoclast-specific or -related DNA sequence. According to a particular embodiment, these cells are capable of replicating the DNA construct comprising the osteoclast-specific or -related DNA, and, optionally, are capable of expressing the osteoclast-specific or -related peptide or gene product encoded by the osteoclast-specific or -related DNA sequence. Also described are antibodies raised against osteoclast-specific or -related gene products, or portions of these gene products, and osteoclast-specific or -related DNA sequences.
The present invention further embraces a method of identifying osteoclast-specific or -related DNA sequences and DNA sequences identified in this manner. In one embodiment, osteoclast-specific or -related cDNA is identified as follows: first, human giant cell tumor of the bone is used to 1) construct a cDNA library; 2) produce 32P-labelled cDNA to use as a stromal cell+, osteoclast+ probe, and 3) produce (by culturing) a stromal cell population lacking osteoclasts. The presence of osteoclasts in the giant cell tumor can be confirmed by histological staining for the osteoclast marker, type 5 tartrate-resistant acid phosphatase (TRAP) and/or with the use of monoclonal antibody reagents.
As described herein, the stromal cell population lacking osteoclasts was produced by dissociating cells of a giant cell tumor, then growing and passaging the cells in tissue culture until the cell population was homogeneous and appeared fibroblastic. The cultured stromal cell population did not contain osteoclasts. The cultured stromal cells were then used to produce a stromal cell+, osteoclastxe2x88x92 32P-labelled cDNA probe.
The cDNA library produced from the giant cell tumor of the bone was then screened in duplicate for hybridization to the cDNA probes: one screen was performed with the giant cell tumor cDNA probe (stromal cell+, osteoclast+), while a duplicate screen was performed using the cultured stromal cell cDNA probe (stromal cell+, osteoclastxe2x88x92). Hybridization to a stromal+, osteoclast+ probe, accompanied by failure to hybridize to a stromal+, osteoclastxe2x88x92 probe indicated that a clone contained nucleic acid sequences specifically expressed by osteoclasts. That is, the clone contained a nucleic acid sequence which is either uniquely expressed by osteoclasts (i.e., osteoclast-specific) or expressed by osteoclasts and select other cells (i.e., osteoclast-related).
In the course of these studies, four clones were identified which contained DNA sequences with significant homology to portions of DNA sequences encoding cysteine proteases, The structural characterization of the coding region cDNA for a particular enzyme, cathepsin X, from which these four sequences originate is also described herein. The present studies also identified one clone which contained a DNA sequence which is a portion of a-DNA sequence encoding a novel human 116-kDa polypeptide subunit of the osteoclast proton pump (OC-116KDa). OC-116KDa mRNA was found at high levels in giant cells of osteoclastomas by Northern analysis but was not detected in tumor stromal cells or in other tissues including kidney, liver, skeletal muscle and brain. OC-116KDa mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization. Thus, it. appears that OC-116kDa represents a novel human 116-kDa subunit of a proton pump which is expressed in osteoclasts in a cell-specific manner,
In another embodiment of the invention, osteoclast-specific or -related genomic DNA is identified through. known hybridization techniques or amplification techniques. This genomic DNA encodes all or a portion of osteoclast-specific or -related peptides or gene products, or encodes all or a portion of the untranslated region of the gene. In one embodiment, the present invention relates to a method of identifying osteoclast-specific or -related DNA by screening a cDNA library or a genomic DNA library with a DNA probe comprising one or more sequences selected from the group consisting of the DNA sequences set out in Table I (SEQ ID NOS: 1-32). Finally, the present invention relates to a nucleotide sequence comprising a DNA sequence selected from the group consisting of the sequences set out in Table I, or their complementary strands, and to peptides or proteins encoded thereby.
The polypeptides and proteins of the present invention have utility as osteodlast cell surface markers. expression of the described polypeptides or proteins is characteristic of osteoclasts, and is unlikely to be found in a wide variety of other cells. Thus, these proteins can be labelled, e.g., radioactively or fluorescently, and used as cell surface markers for osteoclasts.